An important outcome of the recent protein structure initiative is the discovery of numerous protein families that do not form compact rigid structures. These intrinsically unstructured proteins (IUPs) are common in nature and disrupting their function can also result in the onset of certain diseases. We believe that to understand the function of IUPs it is crucial to generate realistic structural ensembles. Such ensembles are difficult to generate for IUPs where experiments predict broad, heterogeneous ensembles of structures that undergo large-scale conformational fluctuations. Experimentally restrained ensembles of loop regions in structured proteins are also difficult to reliably compare to the equilibrium ensemble. We are interested in testing two hypotheses through the use of combined computational and experimental approaches: (i) Experimental data for IUPs which are based on average measurements can be used to generate useful structural ensembles. (ii) These ensembles must be properly weighted in the equilibrium distribution to be useful for understanding protein function. In an effort to expand our understanding of how well experimentally restrained ensembles of unstructured proteins represent the equilibrium ensemble, coarse-graining will be used to generate large ensembles that are restrained using average distance and dihedral angle measurements from nuclear magnetic resonance (NMR) spectroscopy experiments. These structurally diverse ensembles will extend previous work in this area by more thoroughly sampling conformational space. These ensembles will then be re-weighted using non-rigorous methods based on fitting data from small angle x-ray scattering and residual dipolar couplings. We will also extend a rigorous re-weighting approach to loops in structured proteins. These goals will be accomplished through the following Specific Aims: Aim 1: Generate large NMR ensembles for IUPs using coarse-graining. Aim 2: Re-weight NMR ensembles using non-rigorous methods. Aim 3: Extend rigorous re-weighting approach to protein loops. Aim 4: Create and maintain website. The proposed research will give valuable insight into the structure and function of protein loops and IDPs. The resulting software will provide us with a means to distinguish the biologically relevant structures from those that are not relevant. PUBLIC HEALTH RELEVANCE: The proposed research project will give insight into protein structure and function. Thus, our understanding of diseases caused by protein dysfunction will be enhanced.